1. Field of the Invention
The present invention relates to a cellulase preparation and compositions having increased cellulolytic capacity which is useful for increased cellulosic degradation. The invention further relates to a nucleotide sequence of the bgl1 gene encoding extracellular .beta.-glucosidase from Trichoderma reesei, a plasmid vector containing the gene encoding extracellular .beta.-glucosidase and transformant strains with increased copy numbers of the .beta.-glucosidase (bgl1) gene introduced into the genome. More particularly, the present invention relates to a Trichoderma reesei strain that has increased levels of expression of the bgl1 gene resulting in enhanced .beta.-glucosidase protein levels that can be used in conjunction with other compositions to produce a cellulase product having increased cellulolytic capacity.
2. State of the Art
Cellulases are known in the art as enzymes that hydrolyze cellulose (.beta.-1,4-glucan linkages), thereby resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. As noted in "Methods in Enzymology", 160, 25, pages 234 et seq. (1988) and elsewhere, a cellulase system produced by a given microorganism is comprised of several different enzyme components including those identified as exocellobiohydrolases (EC 3.2.1.91) ("CBH"), endoglucanases (EC 3.2.1.4) ("EG"), .beta.-glucosidases (EC 3.2.1.21) ("BG"). Moreover, these classes can be further separated into individual components. For example, multiple CBHs and EGs have been isolated from a variety of bacterial and fungal sources including Trichoderma reesei which contains 2 CBHs, i.e., CBHI and CBHII, and at least 2 EGs, i.e., EGI and EGII components. T. reesei is also classified by some in the literature as T. longibrachiatum. The ratio of CBHI components to EG components (including all of the EG components) in naturally occurring cellulases does not exceed 5:1. For example, see Brown et al., Genetic Control of Environmental Pollutants, Gilbert S. Omenn, Editor, Chapter "Microbial Enzymes and Ligno-Cellulase Utilization", Hollaender Publishing Corp. Variations of this ratio can result from the use of different microorganisms, depending upon the characteristics of the strain, but in any event such ratios do not exceed about 5:1.
The complete cellulase system comprising CBH, EG, and BG is required to efficiently convert crystalline forms of cellulose to glucose. Isolated components are far less effective, if at all, in hydrolyzing crystalline cellulose. Moreover, a synergistic relationship is observed between the cellulase components. That is to say, the effectiveness of the complete/whole system is significantly greater than the sum of the contributions from the isolated components. It has also been suggested by Wood, "Properties of Cellulolytic Systems", Biochem. Soc. Trans., 13, 407-410 (1985), that CBHI and CBHII derived from either T. reesei or P. funiculosum synergistically interact in solubilizing cotton fibers. On the other hand, Shoemaker et al., Bio/Technology, October 1983, disclose that CBHI (derived from T. reesei), by itself, has the highest binding affinity but the lowest specific activity of all forms of cellulose.
The substrate specificity and mode of action of the different cellulase components varies from component to component, which accounts for the synergy of the combined components. For example, the mechanism of cellulose breakdown by cellulase is through the combined action of endo- and exoglucanase activity on crystalline cellulase. It is currently accepted that endoglucanase components act on internal .beta.-1,4-glucosidic bonds in regions of low crystallinity of the cellulose thereby creating chain ends which are recognized by CBH components. The CBH components bind preferentially to the non-reducing end of the cellulose to release cellobiose as the primary product. The more non-reducing chain ends, the greater the action of the CBH components. .beta.-Glucosidase components act primarily to relieve end product inhibition and act on the non-reducing ends of cellooligosaccharides, e.g., cellobiose and cellotriose, to give glucose as the sole product.
.beta.-Glucosidases are essential components of the cellulase system and are important in the complete enzymatic breakdown of cellulose to glucose. The .beta.-glucosidase enzymes can catalyze the hydrolysis of alkyl and/or aryl .beta.-D-glucosides such as methyl .beta.-D-glucoside and p-nitrophenyl glucoside, as well as glycosides containing only carbohydrate residues, such as cellobiose. The catalysis of cellobiose is important since the accumulation of cellobiose inhibits cellobiohydrolases, which in turn inhibits the other cellulase components and thus effects the rate of hydrolysis of cellulose to glucose. This product inhibition by cellobiose is caused by the lack of conversion of cellobiose to glucose. Thus, the .beta.-glucosidases play a key role in effecting the conversion of cellulose to glucose.
Since .beta.-glucosidases can catalyze the hydrolysis of a number of different substrates, the use of this enzyme in a variety of different applications is possible. For instance, some .beta.-glucosidases can be used to liberate aroma in fruit by catalyzing various glucosides present therein. Similarly, some .beta.-glucosidases can hydrolyze grape monoterpenyl .beta.-glucosidase which upon hydrolysis, represents an important potential source of aroma to wine as described by Gunata et al, "Hydrolysis of Grape Monoterpenyl .beta.-D-Glucosides by Various .beta.-Glucosidases", J. Agric. Food Chem., Vol. 38, pp. 1232-1236 (1990).
Furthermore, cellulases can be used in conjunction with yeasts to degrade in biomass cellobiose to glucose that yeasts can further ferment into ethanol. This production of ethanol from readily available sources of cellulose can provide a stable, renewable fuel source. The use of ethanol as a fuel has many advantages compared to petroleum fuel products such as a reduction in urban air pollution, smog, and ozone levels, thus enhancing the environment. Moreover, ethanol as a fuel source would reduce the reliance on foreign oil imports and petrochemical supplies.
But the major drawback to ethanol production from biomass is the lack of .beta.-glucosidase in the system to efficiently convert cellobiose to glucose. Therefore, a cellulase composition that contains an enhanced amount of .beta.-glucosidase would be useful in ethanol production.
.beta.-glucosidases are present in a variety of prokaryotic organisms, as well as eukaryotic organisms. The gene encoding .beta.-glucosidase has been cloned from several prokaryotic organisms and the gene is able to direct the synthesis of detectable amounts of protein in E. coli without requiring extensive genetic engineering, although, in some cases, coupling with a promotor provided by the vector is required. This often is not the case with eukaryotic genes which lack the Shine-Delgarno sequence for prokaryotic translational initiation and which often contain introns. Such genes can sometimes be expressed and detected after transformation of the eukaryotic host S. cerevisia, but many fungal genes fail to be expressed in yeast. Thus, in order to use fungal strains, fungal genes would have to be cloned using methods described herein or by detection with the T. reesei bgl1 gene by nucleic acid hybridization.
The contribution and biochemistry of the .beta.-glucosidase component in cellulose hydrolysis is complicated by the apparent multiplicity of enzyme forms associated with T. reesei (Enari et al, "Purification of Trichoderma reesei and Aspergillus niger .beta.-glucosidase", J. Appl. Biochem., Vol. 3, pp. 157-163 (1981); Umile et al, "A constitutive, plasma membrane bound .beta.-glucosidase in Trichoderma reesei", FEMS Microbiology Letters, Vol. 34, pp. 291-295 (1986); Jackson et al, "Purification and partial characerization of an extracellular .beta.-glucosidase of Trichoderma reesei using cathodic run, polyacrylamide gel electrophoresis", Biotechnol. Bioeng., Vol. 32, pp. 903-909 (1988)). These and many other authors report .beta.-glucosidase enzymes ranging in size from 70-80 Kd and in pI from 7.5-8.5. More recent data suggests that the extracellular and cell wall associated forms of .beta.-glucosidase are the same enzyme (Hofer et al, "A monoclonal antibody against the alkaline extracellular .beta.-glucosidase from Trichoderma reesei: reactivity with other Trichoderma .beta.-glucosidases", Biochim. Biophys. Acta, Vol. 992, pp. 298-306 (1989); Messner and Kubicek, "Evidence for a single, specific .beta.-glucosidase in cell walls from Trichoderma reesei QM9414", Enzyme Microb. Technol., Vol. 12, pp. 685-690 (1990)) and that the variation in size and pI is a result of post translational modification and heterogeneous methods of enzyme purification. It is unknown whether the intracellular .beta.-glucosidase species with a pI of 4.4 and an apparent molecular weight of 98,000 is a novel .beta.-glucosidase (Inglin et al, "Partial purification and characterization of a new intracellular .beta.-glucosidase of Trichoderma reesei", Biochem. J., Vol. 185, pp. 515-519 (1980)) or a proteolytic fragment of the alkaline .beta.-glucosidase associated to another protein (Hofer et al, supra). In addition, since a major part of the detectable .beta.-glucosidase activity remains bound to the cell wall (Kubicek, "Release of carboxymethyl-cellulase and .beta.-glucosidase from cell walls of Trichoderma reesei", Eur. J. Appl. Biotechnol., Vol. 13, pp. 226-231 (1981); Messner and Kubicek, supra; Messner et al, "Isolation of a .beta.-glucosidase binding and activating polysaccharide from cell walls of Trichoderma reesei", Arch. Microbiol., Vol. 154, pp. 150-155 (1990)) commercial preparations of cellulase are thought to be reduced in their ability to produce glucose because of relatively low concentrations of .beta.-glucosidase.
To overcome the problem of .beta.-glucosidase being rate limiting in the production of glucose from cellulose supplementation of the cellulolytic system of Trichoderma reesei with the .beta.-glucosidase of Aspergillus has been attempted with results indicating an increase in rate of saccharification of cellulose to glucose as disclosed by Duff, Biotechnol Letters, 7, 185 (1985). Culturing conditions have also been altered to increase .beta.-glucosidase activity in Trichoderma reesei as illustrated in Sternberg et al, Can. J. Microbiol., 23, 139 (1977) and Tangnu et al, Biotechnol. Bioeng., 23, 1837 (1981), and mutant strains obtained by ultraviolet mutation have been reported to enhance the production of .beta.-glucosidase in Trichoderma reesei. Although these aforementioned methods increase the amount of .beta.-glucosidase in Trichoderma reesei, the methods lack practicality and, in many instances, are not commercially feasible.
A genetically engineered strain of Trichoderma reesei that produces an increased amount of .beta.-glucosidases would be ideal, not only to produce an efficient cellulase system, but to further use the increased levels of expression of the bgl1 gene to produce a cellulase product that has increased cellulolytic capacity. Such a strain can be feasibly produced using transformation.
But, in order to transform mutant strains of Trichoderma reesei, the bgl1 gene must be first characterized in its amino acid sequence, so that it can be cloned to introduce the specific gene into transformed mutant strains of Trichoderma reesei.
Accordingly, it is an object of this invention to characterize the bgl1 gene that encodes for extracellular or cell wall bound .beta.-glucosidase from Trichoderma reesei, to clone the bgl1 gene into a plasmid vector that can be used in the transformation process, and to introduce the bgl1 gene into the Trichoderma reesei genome in multiple copies, which can be used to generate transformed strains with a significant increase in .beta.-glucosidase activity. Moreover, cellulase compositions that contain increased cellulolytic capacity are also disclosed. In yet another aspect of the present invention, the bgl1 gene can be totally deleted from the Trichoderma reesei genome. In addition, altered copies of the bgl1 gene which may change the properties of the enzyme can be reintroduced back into the Trichoderma reesei genome. These and other objects are achieved by the present invention as evidenced by the summary of the invention, description of the preferred embodiments, and claims.